The effect of the dibenzylbutyrolactolic lignan ( )-cubebin on doxorubicin
mutagenicity and recombinogenicity in wing somatic cells of
Drosophila
melanogaster
A.A.A. de Rezende
a, M.L.A. e Silva
b, D.C. Tavares
b, W.R. Cunha
b, K.C.S. Rezende
b, J.K. Bastos
c, M. Lehmann
d,
H.H.R. de Andrade
e, Z.R. Guterres
f, L.P. Silva
g, M.A. Spanó
a,⇑
aUniversidade Federal de Uberlândia, Uberlândia, MG, Brazil bUniversidade de Franca, Franca, SP, Brazil
cUniversidade de São Paulo, Ribeirão Preto, SP, Brazil dUniversidade Luterana do Brasil, Canoas, RS, Brazil
eUniversidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil fUniversidade Estadual do Mato Grosso do Sul, Mundo Novo, MS, Brazil gUniversidade Estadual Paulista Júlio de Mesquita Filho, Assis, SP, Brazil
a r t i c l e
i n f o
Article history:
Received 6 January 2011 Accepted 2 March 2011 Available online 6 March 2011
Keywords:
Somatic Mutation And Recombination Test SMART
Toxicity Cyp6A2
a b s t r a c t
The dibenzylbutyrolactolic lignan ( )-cubebin was isolated from dry seeds ofPiper cubebaL. (Piperaceae). ( )-Cubebin possesses anti-inflammatory, analgesic and antimicrobial activities. Doxorubicin (DXR) is a topoisomerase-interactive agent that may induce single- and double-strand breaks, intercalate into the DNA and generate oxygen free radicals. Here, we examine the mutagenicity and recombinogenicity of dif-ferent concentrations of ( )-cubebin alone or in combination with DXR using standard (ST) and high bio-activation (HB) crosses of the wing Somatic Mutation And Recombination Test inDrosophila melanogaster. The results from both crosses were rather similar. ( )-Cubebin alone did not induce mutation or recom-bination. At lower concentrations, ( )-cubebin statistically reduced the frequencies of DXR-induced mutant spots. At higher concentrations, however, ( )-cubebin was found to potentiate the effects of DXR, leading to either an increase in the production of mutant spots or a reduction, due to toxicity. These results suggest that depending on the concentration, ( )-cubebin may interact with the enzymatic sys-tem that catalyzes the metabolic detoxification of DXR, inhibiting the activity of mitochondrial complex I and thereby scavenging free radicals. Recombination was found to be the major effect of the treatments with DXR alone. The combined treatments reduced DXR mutagenicity but did not affect DXR recombinogenicity.
Ó2011 Elsevier Ltd. All rights reserved.
1. Introduction
Piper cubeba
Linn. (Piperaceae) is a pepper plant that is widely
distributed in tropical and subtropical regions and is popularly
known as
pimenta de Java
(in Brazil),
kemukus
(in Indonesia) or
cu-beb pepper. It is known that extracts from
P. cubeba
have
anti-inflammatory (
Bastos et al., 2001; Choi and Hwang, 2003; Yam
et al., 2008a
), anti-type IV allergic (
Choi and Hwang, 2003
),
antile-ishmanial (
Bodiwala et al., 2007
), genotoxic (
Junqueira et al., 2007
),
antineoplasic (
Yam et al., 2008b
), and molluscicidal activities
(
Pandey and Singh, 2009
).
Lignans are a class of secondary plant metabolites that are
pro-duced by the oxidative dimerization of two phenylpropanoid units.
Interest in lignans and their synthetic derivatives has grown due to
their applications for cancer chemotherapy and their various other
pharmacological effects (
Saleem et al., 2005
). Here, ( )-cubebin, a
dibenzylbutyrolactolic lignan, was isolated from the dry seeds of
P. cubeba
L. This lignan is known to possess anti-inflammatory
(
Bastos et al., 2001; da Silva et al., 2005
), analgesic (
da Silva
et al., 2005
), and antimicrobial activities (
Silva et al., 2007, 2009
).
Although some ( )-cubebin derivative compounds (( )-hinokinin;
( )-O-benzyl cubebin; ( )-O-(N,N-dimethylamino-ethyl)-cubebin)
inhibit the free amastigote forms of
Trypanosoma cruzi, the natural
( )-cubebin molecule itself, which is used as the starting
com-pound to obtain the evaluated dibenzylbutyrolactolic derivatives,
does not affect the growth of trypomastigote forms of
T. cruzi
(
de
Souza et al., 2005
). ( )-Cubebin and derivatives isolated from
P.
cubeba
were found to significantly inhibit cytochrome P450
(CYP3A4) (
Usia et al., 2005, 2006
) and the NADH oxidase activity
of mitochondrial complex I (
Saraiva et al., 2009
).
0278-6915/$ - see front matterÓ2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.001
⇑
Corresponding author. Tel.: +55 34 32182505; fax: +55 34 32182203.E-mail address:maspano@ufu.br(M.A. Spanó).
Contents lists available at
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Food and Chemical Toxicology
The anthracycline antibiotic Doxorubicin (DXR), a drug that
tar-gets topoisomerase II (Top2) (
Islaih et al., 2005
), is one of the most
effective anticancer drugs used in the clinic (
Lyu et al., 2007
). This
drug may induce mutations by intercalating formaldehyde adducts
in the DNA (
Spencer et al., 2008
) or by inducing the formation of
oxygen free radicals (
Navarro et al., 2006
), single- and
double-strand DNA breaks (
Lyu et al., 2007
) and somatic recombination
(
Lehmann et al., 2003; Valadares et al., 2008; de Rezende et al.,
2009; Sousa et al., 2009
).
The wing Somatic Mutation And Recombination Test (SMART)
using
Drosophila melanogaster
was developed to detect the loss of
heterozygosity of suitable gene markers that have detectable
phe-notypes that are expressed on the wings. This assay is an efficient
and quick method for quantitating the recombinogenic and
muta-genic potential of chemical and physical agents (
Graf et al., 1996;
Vogel et al., 1999; Spanó et al., 2001
). For this purpose, two crosses
are typically used: the standard (ST) cross (
Graf et al., 1989
) and a
high bioactivation (HB) cross (
Graf and van Schaik, 1992
). The ST
cross uses strains carrying basal levels of the metabolizing
cyto-chrome P450 enzyme (Cyp6A2) and is used to detect direct-acting
genotoxins. The HB cross uses strains with high levels of Cyp6A2
and is used to detect indirect-acting genotoxins that exert their
genotoxic activity only when metabolized (
Frölich and Würgler,
1989; Graf and van Schaik, 1992; Saner et al., 1996
).
Several reports have demonstrated the probability of
pharma-cokinetic interaction between natural compounds and herbal
prod-ucts with conventional drugs when they are administered
simultaneously (
Usia et al., 2006
). Here, we examine the
mutage-nicity and recombinogemutage-nicity of the dibenzylbutyrolactolic lignan
( )-cubebin when administered alone or simultaneously with
DXR.
2. Materials and methods
2.1. Chemical compounds and media
( )-Cubebin was isolated and purified from the seeds ofP. cubebaL. as previ-ously described (Silva et al., 2007). Doxorubicin (DXR) (RubidoxÒ
– Laboratório Químico Farmacêutico Bergamo Ltda., Taboão da Serra, SP, Brazil) (CAS 23214– 92-8) was obtained from the Hospital de Clínicas da Universidade Federal de Uber-lândia, MG, Brazil. Ultrapure water (18.2XX) was obtained from a MilliQ system (Millipore, Vimodrone, Milan, Italy). ( )-Cubebin was dissolved in a mixture of 1% Tween 80 (Fluka, Buchs, Switzerland) and 3% ethanol (Neon, São Paulo, Brazil) in ultrapure water. DXR was dissolved in ultrapure water. The solutions were al-ways prepared immediately before use. As an alternative medium, instant mashed potato flakes (YokiÒ
Alimentos S. A., São Bernardo do Campo, SP, Brazil) were used. The structural formula of ( )-cubebin is shown inFig. 1.
2.2. The Somatic Mutation And Recombination Test (SMART)
Two crosses were carried out to produce the experimental larval progeny. The first was the standard (ST) cross, in which virgin females of strainflr3/In(3LR)TM3,
ri pp sep l(3)89Aa bx34ee BdSwere crossed withmwh/mwhmales (Graf et al.,
1984, 1989). The second was the high bioactivation (HB) cross, in which virgin fe-males of strainORR/ORR;flr3/In(3LR)TM3, ri ppsep l(3)89Aa bx34ee BdSwere crossed
withmwh/mwhmales (Graf and van Schaik, 1992). Each cross produced marker-heterozygous (MH) flies (mwh flr+/mwh+flr3) with normal wings and
balancer-heterozygous (BH) flies (mwh flr+/mwh+TM3,BdS) with serrated wings. From both
crosses, eggs were collected for 8 h in culture glass bottles with an agar-agar base
(4% w/v) topped with a thick layer of live baker’s yeast supplemented with sucrose. After 72 h (± 4 h), third instar larvae were washed out of the bottles with ultrapure water (MilliQ system) and collected in a stainless steel strainer. For chronic feeding, four sets of vials for each cross were prepared with 1.5 g of mashed potato flakes (YokiÒ
Alimentos S.A., Brazil) and 5 ml of a solution containing ( )-cubebin alone (at a final concentration of 0.25, 0.5, 1.0, 2.0 or 4.0 mM) or with DXR (0.2 mM). Neg-ative (1% Tween-80 and 3% ethanol in distilled water) and positive (DXR 0.2 mM) controls were included in both experiments. The experiments were conducted at 25 ± 1°C and approximately 60% humidity. The larvae were counted before the dis-tribution in two series of these vials. The number of hatched flies was used to cal-culate the survival rates upon exposure. From the other two sets of vials, the hatched flies were stored in 70% (v/v) ethanol. The wings were removed and mounted on slides with Faure’s solution (gum arabic 30 g, glycerol 20 ml, chloral hydrate 50 g and distilled water 50 ml) and analyzed for spots under a compound microscope at 40x magnification. Frequency and size of single and twin spots were recorded. Single spots (mwhorflr3) can result from mutational events, chromosome
aberrations or mitotic recombination (crossing over between the two marker genes). Twin spots (mwhandflr3) are produced exclusively by mitotic
recombina-tion (crossing over between the markerflr3and the centromere of chromosome
3). The wings of BH flies were mounted and analyzed after verifying that positive responses were obtained in the MH progeny. In the wings of BH flies, onlymwh sin-gle spots can be recovered. These spots are due to only mutational events because recombination is suppressed in inversion-heterozygous cells with the multiply in-verted TM3 balancer chromosome (Graf et al., 1984; Guzmán-Rincón and Graf, 1995).
2.3. Statistical analysis
For each treated series, 50 flies of both sexes were scored. The multiple-decision procedure (Frei and Wurgler, 1988) was used to analyze the data, resulting in three different diagnoses: negative, positive or inconclusive. The frequency of each type of spot (small single, large single or twin) and the total frequency of spots per fly for each treatment were compared pair-wise (i.e., negative control versus ( )-cubebin; positive control (DXR) alone versus DXR plus ( )-cubebin) according to Kastenbaum and Bowman (1970)withp= 0.05 (Frei and Wurgler, 1988, 1995). All inconclusive and weak results were analyzed with the non-parametric U-test of Mann, Whitney and Wilcoxon (a = b = 0.05, one sided) (Frei and Wurgler, 1995). Based on the clone induction frequency per 105cells, the recombinogenic
activity was calculated as follows. Frequency of mutation (FM) = frequency of clones in BH flies/frequency of clones in MH flies. Frequency of recombination (FR) = 1 – frequency of mutation (FM). Frequencies of total spots (FT) = total spots observed in MH flies (consideringmwhandflr3 spots)/number of flies (Santos
et al., 1999; Sinigaglia et al., 2004, 2006). Based on the control-corrected spot fre-quencies per 105cells, the percentage of ( )-cubebin inhibition was calculated
as: (DXR alone – ( )-cubebin plus DXR/DXR alone) – 100 (Abraham, 1994). Statis-tical comparisons of survival rates were made with the Chi-squared test for ratios of independent samples.
3. Results
Third instar larvae of
D. melanogaster
obtained from both (ST
and HB) crosses were fed chronically (approximately 48 h) with
( )-cubebin (0.25, 0.5, 1.0, 2.0 or 4.0 mM) alone or in combination
with DXR (0.2 mM). Each treatment was done in duplicate. The
data were pooled after verifying that there were no significant
dif-ferences between repetitions. The concentrations were chosen
based on a dose response test, for which the survival rates of flies
are given in
Table 1
. Although 4.0 mM ( )-cubebin alone
signifi-cantly decreases the survival rates, this concentration was also
tested in association with DXR.
At concentrations below 2.0 mM, ( )-cubebin was not found to
be toxic. Thus, we found it particularly important to evaluate the
modulatory effects of low concentrations on DNA damage induced
by DXR in somatic cells of
D. melanogaster.
The highest
concentra-tion (4.0 mM) of ( )-cubebin was found to be toxic in the ST cross,
when administered alone or combination with DXR. A significant
decrease in survival rates relative to the negative control group
(ultrapure water) was observed. This concentration was also found
to be toxic in the HB cross when administered with DXR.
The results of the ST cross of the SMART assays using
D.
melanogaster
are depicted in
Table 2
. In the MH individuals,
( )-cubebin alone did not show any genotoxicity at the doses
used. The DXR treatment, as expected, induced positive results
for all categories of spots when compared to the negative
O
O
O
O
O
HO
control. Lower concentrations of ( )-cubebin (0.25, 0.5 or
1.0 mM) when administered with DXR (0.2 mM) were found to
inhibit DXR-induced DNA damage (54.66, 26.21 and 26.84%,
respectively). The simultaneous administration of DXR with
( )-cubebin (2.0 mM), however, was found to significantly
increase the number of DXR-induced wing spots (by 94.30%).
The wings of the BH flies resulting from the simultaneous
appli-cation of both drugs were also mounted and scored. This procedure
enabled us to quantify the contribution of mutagenic and
recombi-nogenic events to the final genotoxicity observed (
Frei et al., 1992;
Graf et al., 1992
).
In the BH individuals of the ST cross, DXR (0.2 mM) induced a
significant increase in the mutant spot frequency relative to the
negative control. In the combined treatments, only 0.5 mM (
)-cubebin significantly reduced the total number of spots induced
by DXR. The other concentrations of ( )-cubebin (0.25, 1.0, 2.0
and 4.0 mM) did not affect DXR mutagenicity.
Based on the clone induction frequency per 10
5cells, we
com-pared the number of observed spots in the MH and BH flies and
quantified the contribution (%) of mutation and recombination to
the total number of observed spots (
Frei et al., 1992; Graf et al.,
1992; Abraham, 1994
). We found that the induced spots were
mainly due to recombination. We also found that ( )-cubebin does
not affect the recombinogenic activity of DXR (
Fig. 2
).
The results of the HB cross of the SMART assays with
D.
melano-gaster
are summarized in
Table 3
. The results obtained with the
MH individuals of the HB cross treated with ( )-cubebin alone
were negative at all tested concentrations. DXR statistically
increased all categories of spots when compared to the negative
control. When administered with DXR, all concentrations of (
)-cubebin were found to statistically inhibit DXR-induced DNA
damage, although the inhibition have not been dose-related.
In the BH individuals, DXR induced a significant increase in
mu-tant spots, relative to the negative control. Compared to DXR alone,
the treatments of 0.25, 1.0 or 4.0 mM ( )-cubebin administered
with DXR produced significantly fewer mutant spots, while 0.5
and 2.0 mM ( )-cubebin had no effect on the production of mutant
spots.
Table 1
Survival rates upon exposure to different concentrations of ( )-cubebin alone or in combination with doxorubicin relative to control groups (ultrapure water and doxorubicin) in the wing Somatic Mutation And Recombination Test inD. melanogaster.
Compounds Standard cross High bioactivation cross
DXR (mM) Cubebin (mM) Survival (%) p -value
Survival (%) p -value
0 0 95 95
0 0.25 90 >0.05 90 >0.05
0 0.5 100 >0.05 90 >0.05
0 1.0 95 >0.05 90 >0.05
0 2.0 85 >0.05 90 >0.05
0 4.0 60 <0.05 90 >0.05
0.2 0 95 >0.05 95 >0.05
0.2 0.25 85 >0.05 80 >0.05
0.2 0.5 85 >0.05 80 >0.05
0.2 1.0 85 >0.05 80 >0.05
0.2 2.0 75 >0.05 70 >0.05
0.2 4.0 50 <0.05 65 <0.05
Statistical comparisons of survival rates were made with the Chi-squared test for ratios of independent samples.
Table 2
Summary of results obtained with theDrosophilawing spot test (SMART) in the marker-heterozygous (MH) and balancer-heterozygous (BH) progeny of the standard cross (ST) after chronic treatment of larvae with ( )-cubebin and Doxorubicin (DXR).
Genotypes and treatments
Number of flies
Spots per fly (number of spots) statistical diagnosisa Spots with
mwhclonec
Frequency of clone formation/105cells
per cell divisiond
Recombination (%)
Inhibitione (;) or inductione (")(%) Small single
spots (1–2 cells)b
Large single spots (>2 cells) b
Twin spots Total spots DXR
(mM) ( )-Cubebin (mM)
Observed Control Corrected
mwh/flr3
0 0 50 0.32 (16) 0.06 (03) 0.02 (01) 0.40 (20) 20 0.82
0 0.25 50 0.16 (08) 0.00 (00) 0.00 (00) 0.16 (08) 08 0.33 0.49 0 0.50 50 0.16 (08) 0.00 (00) 0.00 (00) 0.16 (08) 08 0.33 0.49 0 1.00 50 0.14 (07) 0.04 (02) 0.00 (00) 0.18 (09) 09 0.37 0.45 0 2.00 50 0.16 (08) 0.08 (04) 0.02 (01) 0.26 (13) 13 0.53 0.29 0 4.00 50 0.22 (11) 0.02 (01) 0.00 (00) 0.24 (12) 12 0.49 0.33
0.20 0 50 2.78 (139)+ 2.38 (119)+ 2.66 (133)+ 7.72 (386)+ 371 15.20 14.38 91.90
0.20 0.25 50 1.20 (60)* 0.92 (46)* 1.56 (78)* 3.68 (184)* 179 7.34 6.52 86.02 54.66;
0.20 0.50 50 1.52 (76)* 1.58 (79)* 2.56 (128) 5.66 (283)* 279 11.43 10.61 94.27 26.21;
0.20 1.00 50 1.40 (70)* 1.46 (73)* 2.80 (140) 5.66 (283)* 277 11.34 10.52 88.10 26.84;
0.20 2.00 50 3.42 (171)* 4.08 (204)* 6.92 (346)* 14.42 (721)* 702 28.76 27.94 94.59 94.30"
0.20 4.00 50 2.62 (131) 2.04 (102) 2.72 (136) 7.38 (369) 354 14.20 13.38 94.10 06.94
mwh/TM3
0 0 50 0.26 (13) 0.00 (00) f 0.26 (13) 13 0.53
0.20 0 50 0.50 (25)+ 0.10 (05)+ 0.60 (30)+ 30 1.23 0.70
0.20 0.25 50 0.40 (20) 0.10 (05) 0.50 (25) 25 1.01 0.48
0.20 0.50 50 0.08 (04)* 0.24 (12) 0.32 (16)* 16 0.64 0.11
0.20 1.00 50 0.40 (20) 0.26 (13)* 0.66 (33) 33 1.34 0.81
0.20 2.00 50 0.66 (33) 0.10 (05) 0.76 (38) 38 1.56 1.03
0.20 4.00 50 0.42 (21) 0.00 (00)* 0.42 (21) 21 0.85 0.32
Marker-trans-heterozygous flies (mwh/flr3) and balancer-heterozygous flies (mwh/TM3) were evaluated.
*P60.05 vs. DXR only.
a Statistical diagnoses according toFrei and Wurgler (1988, 1995).U-test, two sided; probability levels: , negative; +, positive; i, inconclusive;P60.05 vs. untreated
control;
b Including rareflr3single spots.
c Consideringmwhclones frommwhsingle and twin spots.
d Frequency of clone formation: clones/flies/48,800 cells (without size correction). e Calculated as{[DXR alone – DXR + ( )-cubebin] /DXR}100, according toAbraham (1994).
f Balancer chromosomeTM3does not carry theflr3mutation and recombination is suppressed, due to the multiple inverted regions in these chromosomes.
By comparing the number of observed spots in the MH flies and
BH flies, we once again found that the induced spots were mainly
due to recombination. We found that ( )-cubebin displays only
anti-mutagenic activity and does not interfere with DXR
recombi-nogenicity (
Fig. 2
).
4. Discussion
The mutagenic and/or recombinogenic potential of ( )-cubebin,
alone and in combination with the chemotherapeutic agent DXR,
was assayed using two crosses (ST and HB) of the wing Somatic
Mutation And Recombination Test (SMART) in
D. melanogaster.
We obtained similar data in both crosses and found that (
)-cube-bin alone does not modify the frequency of spontaneous mutant
spots in this test system.
The reference mutagen DXR significantly increased all
catego-ries of spots. Previous studies using the SMART assay have shown
that the major mutational contribution of DXR is its ability to
in-duce recombination (
Lehmann et al., 2003; Valadares et al., 2008;
de Rezende et al., 2009; Sousa et al., 2009
). DXR is a
chemothera-peutic agent that induces single- and double-stranded DNA breaks,
which are processed by recombinational DNA repair pathways.
DNA double-strand breaks (DSBs) are a serious threat to the cell
and can lead to chromosomal aberration, mutation and cancer.
DSBs in human cells are repaired via non-homologous DNA end
joining and homologous recombination repair pathways (
Poplaw-ski et al., 2010
). Homologous recombination can result in a loss
of heterozygosity or genetic rearrangements. Some of these genetic
alterations can be correlated with the manifestation of recessive
heritable diseases and may play a primary role in carcinogenesis.
More likely, however, they are probably involved in secondary
and subsequent steps of carcinogenesis that reveal recessive
onco-genic mutations (
Bishop and Schiestl, 2002, 2003
).
There is an increasing amount of evidence to suggest that
can-cer and other mutation-related diseases can be prevented not only
by limiting exposure to recognized risk factors but also with the
in-take of protective factors and by modulating the defense
mecha-nisms of the host organism. This strategy, referred to as
chemoprevention, can be pursued either with suitable
pharmaco-logical agents and/or by dietary factors (
Ferguson et al., 2005
).
Here, we examined the mutagenicity and recombinogenicity of
the dibenzylbutyrolactolic lignan ( )-cubebin extracted from
seeds of
P. cubeba
L. when administered alone or simultaneously
with the pharmacological agent DXR.
The association of ( )-cubebin with DXR in the ST cross was
found to produce different results depending on the concentration
used. At lower concentrations (0.25, 0.5 or 1.0 mM) ( )-cubebin
significantly reduces the frequency of DXR-induced mutant spots.
At 2.0 mM, ( )-cubebin strongly increases DXR-induced
mutage-nicity and recombinogemutage-nicity, affecting all types of spots (small
single, large single and twin spots). The magnitude of the
comu-tagenicity was found to be considerable, leading to an
enhance-ment of 94.30%. At 4.0 mM, ( )-cubebin slightly reduces the
frequency of DXR-induced DNA damage. Given the significant
de-crease in survival rates that was observed in flies treated with
4.0 mM ( )-cubebin (
Table 1
), we think that the tendency of
reduction in DXR-induced DNA damage can be attributed to
cyto-toxicity, rather than a protective effect of ( )-cubebin.
In the HB cross, all concentrations of ( )-cubebin were found to
significantly reduce the frequency of DXR-induced mutant spots.
Table 3
Summary of results obtained with theDrosophilawing spot test (SMART) in the marker-heterozygous (MH) and balancer-heterozygous (BH) progeny of the high bioactivation cross (HB) after chronic treatment of larvae with ( )-cubebin and Doxorubicin (DXR).
Genotypes and treatments
Number of flies
Spots per fly (number of spots) statistical diagnosisa Spots with
mwhclonec
Frequency of clone formation/105cells
per cell divisiond
Recombination (%)
Inhibitione (%) Small single
spots (1–2 cells)b
Large single spots (>2 cells)b
Twin spots Total spots DXR
(mM) ( )-Cubebin (mM)
Observed Control Corrected
mwh/flr3
0 0 50 0.38 (19) 0.08 (04) 0.08 (04) 0.54 (27) 27 1.11
0 0.25 50 0.28 (14) 0.08 (04) 0.02 (01) 0.38 (19) 19 0.78 0.33 0 0.50 50 0.26 (13) 0.02 (01) 0.04 (02) 0.32 (16) 16 0.66 0.45 0 1.00 50 0.40 (20) 0.08 (04) 0.00 (00) 0.48 (24) 24 0.98 0.13 0 2.00 50 0.40 (20) 0.04 (02) 0.00 (00) 0.44 (22) 22 0.90 0.21 0 4.00 50 0.44 (22) 0.16 (08) 0.02 (01) 0.62 (31) 30 1.22 0.10
0.20 0 50 2.80 (140)+ 2.78 (139)+ 3.20 (160)+ 8.78 (439)+ 420 17.21 16.10 90.89
0.20 0.25 50 1.74 (87)* 1.50 (75)* 1.66 (83)* 4.90 (245)* 245 10.04 8.93 92.62 44.54
0.20 0.50 50 2.32 (116) 1.46 (73)* 2.30 (115)* 6.08 (304)* 304 12.46 11.35 87.38 29.50
0.20 1.00 50 2.54 (127) 2.18 (109)* 2.64 (132) 7.36 (368)* 363 14.88 13.77 93.11 14.47
0.20 2.00 50 2.28 (114) 1.64 (82)* 2.16 (108)* 6.08 (304)* 301 12.34 11.23 88.48 30.25
0.20 4.00 50 2.20 (110)* 1.20 (60)* 2.34 (117)* 5.74 (287)* 285 11.67 10.56 89.79 34.41
mwh/TM3
0 0 50 0.26 (13) 0.00 (00) f 0.26 (13) 13 0.53
0.20 0 50 0.50 (25)+ 0.30 (15)+ 0.80 (40)+ 40 1.64 1.11
0.20 0.25 50 0.50 (25) 0.00 (05) 0.50 (25)* 25 1.02 0.49
0.20 0.50 50 0.70 (35) 0.00 (00) 0.70 (35) 35 1.42 0.89
0.20 1.00 50 0.50 (25) 0.00 (00)* 0.66 (25)* 25 1.02 0.49
0.20 2.00 50 0.66 (33) 0.10 (05)* 0.76 (38) 38 1.56 1.03
0.20 4.00 50 0.42 (21) 0.00 (00)* 0.42 (21)* 21 0.85 0.32
Marker-trans-heterozygous flies (mwh/flr3) and balancer-heterozygous flies (mwh/TM3) were evaluated.
*P60.05 vs. DXR only.
aStatistical diagnoses according toFrei and Wurgler (1988, 1995).U-test, two sided; probability levels: , negative; +, positive; i, inconclusive;P60.05 vs. untreated
control;
b Including rareflr3single spots.
c Consideringmwhclones frommwhsingle and twin spots.
d Frequency of clone formation: clones/flies/48,800 cells (without size correction). eCalculated as{[DXR alone – DXR + ( )-cubebin] /DXR}100, according toAbraham (1994).
However, given the significant decrease in survival rates observed
in the ST cross (
Table 1
), we conclude that the reduction in
DXR-induced mutant spots that can be observed upon treatment with
0.2 or 0.4 mM ( )-cubebin in the HB cross could be due to
cytotoxicity.
Similar data have been generated for ( )-hinokinin (HK), a
dib-enzylbutyrolactolic lignan that is obtained by from ( )-cubebin,
using the micronucleus (MN) test in V79 Chinese hamster lung
fibroblasts to assay the effect on DXR clastogenicity. At lower
con-centrations, HK significantly protects against DXR-induced MN
for-mation. This effect is thought to be due to of the ability of HK to
quench reactive oxygen species. At higher concentrations, HK
potentiates DXR-induced clastogenicity, suggesting that higher
doses of HK may increase the oxidative stress generated by DXR
(
Resende et al., 2010
).
The cytochrome P450 (CYP) superfamily is the most important
drug-metabolizing enzyme group that activates promutagens
(
Takiguchi et al., 2010
). Previous studies have shown that an
ab-sence of mutagenicity may result from the metabolic deactivation
of a compound. For example, nor-nitrogen mustard (NNM) was
found to be non-mutagenic when fed to adult flies in a sex-linked
recessive lethal test. In combination with 1-phenylimidazole (PhI),
an inhibitor of cytochrome P-450, sufficient quantities of the
muta-gen was able to reach the gonads and produce significant muta-genetic
damage. These data suggest that NNM is deactivated by this family
of cytochrome P-450 (
Zijlstra and Vogel, 1988
). It has been
pro-posed that a similar mechanism can explain the significant
poten-tiating action of tannic acid when administered simultaneously
with either of the alkylating agents methylmethanesulfonate or
nitrogen mustard in a SMART assay in
D. melanogaster
(
Lehmann
et al., 2000
).
Previous studies have shown that MeOH- and EtOAc-soluble
fractions of
P. cubeba
significantly inhibit the human
CYP3A4-med-iated metabolism of (N-methyl-
14C)erythromycin (
Usia et al.,
2006
). Thus, it is possible that ( )-cubebin acts by interacting with
the enzyme systems that catalyze the metabolic detoxification of
DXR in
D. melanogaster. Such an interaction could explain the high
frequency of mutant spots observed at 2.0 mM (ST cross) and the
Fig. 2.Contribution of recombination and mutation (in percentage) to total mwh wing spot induction observed in MH individuals from the ST and HB crosses treated with DXR alone and in combination with different concentrations of ( )-cubebin.
cytotoxic effect observed at 2.0 mM (HB cross) and 4.0 mM (in both
ST and HB crosses).
Mitochondria have also been often implicated in cell death. (
)-Cubebin and its derivatives typically inhibit the mitochondrial
complex I, as demonstrated by the inhibition of
glutamate/ma-late-supported state 3 respiration of mitochondria and the NADH
oxidase activity of submitochondrial particles (
Saraiva et al.,
2009
). The inhibition of mitochondrial complex I is another
possi-ble mechanism to explain the toxicity observed at high (
)-cubebin concentrations.
In the
Drosophila
SMART assay ( )-cubebin significantly alters
the generation of DXR-induced wing spots, which are the result
of somatic mutation and mitotic recombination. Our data, in
bination with the data from previous studies using different
com-pounds, allow us to suggest that ( )-cubebin may act as a free
radical scavenger at low concentrations and a pro-oxidant at
high-er concentrations when it inthigh-eracts with the enzymatic system that
catalyzes the metabolic detoxification of DXR. Alternatively, (
)-cubebin may inhibit mitochondrial complex I, leading to cell death.
In this study, we showed that ( )-cubebin can positively or
neg-atively influence DXR-induced mutagenicity, depending on the
concentration. With the long-term goal of developing
chemopre-vention strategies, future work will focus on investigating the
con-ditions under which ( )-cubebin can prevent genome damage, its
mechanisms of action and its pharmacokinetic interaction with
conventional drugs.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by grants from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),
Fun-dação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),
Uni-versidade de Franca (UNIFRAN) and UniUni-versidade Federal de
Uberlândia (UFU). Alexandre Azenha Alves de Rezende was
sup-ported by a fellowship from Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES).
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